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Two Novel Mutations in the Thyrotropin (TSH) Receptor
Gene in a Child with Resistance to TSH*
R. J. CLIFTON-BLIGH†, J. W. GREGORY, M. LUDGATE, R. JOHN, L. PERSANI,
C. ASTERIA, P. BECK-PECCOZ, AND V. K. K. CHATTERJEE
Department of Medicine, University of Cambridge, Addenbrooke’s Hospital (R.J.C.-B., V.K.K.C.),
Cambridge, United Kingdom CB2 2QQ; the Departments of Child Health (J.W.G.), Pathology (M.L.),
and Medical Biochemistry (R.J.), University Hospital of Wales, Cardiff, United Kingdom CF4 4XW;
and the Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore, IRCCS and Centro
Auxologico Italiano IRCCS (L.P., C.A., P.B.-P.), Milan, Italy
ABSTRACT
The TSH receptor is a G protein-coupled receptor that mediates the
effects of TSH in thyroid development, growth, and synthetic func-
tion. We report here that a child with features of TSH resistance,
including markedly increased serum TSH concentrations and low
normal thyroid hormone levels, is a compound heterozygote for two
novel mutations in the TSH receptor gene. One allele hasaGtoA
transition corresponding to an arginine to glutamine change at codon
109 (R109Q) in the extracellular domain of the receptor. The other
allele hasaGtoAtransition corresponding to a premature termi-
nation codon at tryptophan 546 (W546X) in the fourth transmem-
brane segment. Each parent is heterozygous for one mutation, and
both parents have normal thyroid function. Cells transiently trans-
fected with the R109Q mutant exhibited reduced membrane binding
of [
125
I]TSH and impaired signal transduction in response to TSH. In
contrast, the W546X mutant was nonfunctional, with negligible mem-
brane radioligand binding. Our findings indicate that a single normal
TSH receptor allele is sufficient for normal thyroid function, but that
the compound abnormality in the proband leads to TSH resistance.
(J Clin Endocrinol Metab 82: 1094–1100, 1997)
P
ITUITARY TSH is critical for thyroid development and
function, and its actions are mediated by a transmem-
brane receptor, which, together with LH/CG and FSH re-
ceptors, belongs to a subfamily of G protein-coupled recep-
tors. The principal biological effects of TSH on the thyrocyte
occur by receptor-mediated activation of G
s
a
and subse-
quent generation of intracellular cAMP (1, 2). The TSH re-
ceptor gene is located on chromosome 14 (3, 4), and the
extracellular domain is encoded by nine exons, with exon 10
coding transmembrane and intracellular portions of the re-
ceptor. In keeping with many other G protein-coupled re-
ceptors (5, 6), both gain and loss of function mutations have
been described in the TSH receptor. Gain of function muta-
tions are autosomal dominant and, when somatic, cause thy-
roid hyperfunctioning adenomas (7–13), whereas germline
inheritance leads to nonautoimmune congenital hyperthy-
roidism (14–18). Autosomal recessive inheritance of TSH
resistance caused by mutations in the TSH receptor was first
reported in three siblings who were compound heterozy-
gotes for mutations of two extracellular domain residues
(19). All three patients were euthyroid with normal serum
thyroid hormone concentrations but increased serum con-
centrations of TSH, indicating that the resistance in these
cases was partial. Each parent was heterozygous for one of
these mutations.
We describe a child with greatly increased serum TSH
concentrations and low normal thyroid hormone concentra-
tions who is a compound heterozygote for two novel mu-
tations in the TSH receptor gene, one inherited from each
parent. The mutation inherited from the mother corresponds
to a TSH receptor truncated in the fourth transmembrane
domain that is functionally inactive in vitro. The paternal
mutation lies in the extracellular segment of the receptor and
has a reduced binding affinity for TSH, resulting in impaired
signal transduction.
Case Report
The patient wasidentified after a positive resulton neonatal screening
for hypothyroidism. He is the only child of unrelated parents. He was
delivered by caesarean section at 38 weeks gestation, weighed 2.45 kg,
and had transienthypoglycemia. Thyroid function tests at 8 weeks of age
showed a serum free T
4
concentration of 10 pmol/L (normal range,
12–28 pmol/L) with a TSH of 92 mU/L (normal range, 0.4–4.0 mU/L).
There were no clinical features of hypothyroidism. He was commenced
on T
4
. The progress of his thyroid function tests in response to T
4
administration is shown in Fig. 1. At 12 months of age, a T
4
dose of 60
m
g suppressed serum TSH into the normal range but with raised serum
free T
4
concentrations of 40 pmol/L. While on treatment, his mother
described him as irritable; when treatment was discontinued at 2 yr of
age, his behavior became more normal. After birth, he demonstrated
catch-up growth to the 50th percentile, and thereafter his growth, bone
maturation, and development have continued to be normal without
treatment. His hearing is normal, as measured by otoacoustic emissions.
A thyroid isotope scan at 2 yr of age showed a gland of normal size and
location, with uniform tracer uptake. Thyroid ultrasonography was
normal. A perchlorate discharge test was slightly elevated at 15%. Both
patient and mother were negative for thyroid autoantibodies. Serum
electrolyte, GH, PRL, LH, FSH, PTH, and calcium concentrations were
all normal in the patient. He remains clinically euthyroid without ther-
Received November 7, 1996. Revision received December 9, 1996.
Accepted December 30, 1996.
Address all correspondence and requests for reprints to: Dr. V. K. K.
Chatterjee, Department of Medicine, University of Cambridge, Level 5,
Addenbrooke’s Hospital, Hills Road, Cambridge, United Kingdom CB2
2QQ.
* This work was supported by the Wellcome Trust (to V.K.K.C.), the
Medical Research Council (to M.L.), and Murst and CNR, Rome (to
P.B.P.).
† Commonwealth Foundation Research Scholar.
0021-972X/97/$03.00/0 Vol. 82, No. 4
Journal of Clinical Endocrinology and Metabolism Printed in U.S.A.
Copyright © 1997 by The Endocrine Society
1094
apy. His most recent thyroid function tests at 3 yr of age show a TSH
of 134 mU/L and free T
4
of 12 pmol/L. The results of a T
3
suppression
test are shown in Table 2. At this time the serum glycoprotein hormone
a
-subunit level was 2.5 ng/mL (normal range, 0.24–1.05 ng/mL), but the
molar ratio of
a
-subunit to TSH was normal at 0.17 (normal range, ,5.5).
TRH tests in both parents were normal (data not shown).
Materials and Methods
Genetic analyses
Patient lymphocytes were Epstein-Barr virus-transformed (ECACC,
Porton Down, Salisbury) and grown in RPMI 1640 supplemented with
10% FCS. Ribonucleic acid and genomic DNA were extracted from the
lymphocytes using TRIzol according to the manufacturer’s instructions
(Life Technologies, Paisley, Scotland). First strand complementary DNA
(cDNA) was synthesized using Superscript II reverse transcriptase (Life
Technologies), and nested reverse transcription-PCR was subsequently
performed with primers spanning the extracellular and transmembrane
coding regions of the TSH receptor. Genomic DNA was isolated from
peripheral blood leukocytes from each parent using standard tech-
niques. Exons of the TSH receptor were amplified from patient and
parents by PCR using recently published intronic primer sequences (20).
The forward primer in each case was tagged with the universal M13
primer sequence. Direct sequencing of the PCR products was under-
taken by cycle sequencing using dye-labeled universal 21M13 primers
(Applied Biosystems/Perkin-Elmer, Cheshire, UK) and analyzed by
electrophoresis on an ABI 373 sequencer (Applied Biosystems, Foster
City, CA). Primer sequences and PCR conditions are available on
request.
Functional studies
TSH bioactivity was measured in Chinese hamster ovary cells ex-
pressing recombinant human TSH receptor as previously described (21).
DNA fragments bearing each mutation were replaced in full-length
wild-type TSH receptor cDNA cloned into the eukaryotic expression
vector pSVL (22), and constructs were verified by sequencing. JEG3
(human choriocarcinoma) cells were grown in Optimem (Life Technol-
ogies) supplemented with 2% (vol/vol) FCS and 1% (vol/vol) penicillin,
streptomycin, fungizone (Life Technologies). Eighteen hours before
transfection, the medium was replaced with Optimem containing 2%
resin-stripped FCS. Cells were transfected by a 5-h exposure to calcium
phosphate containing
a
LUC reporter, TSH receptor expression vector,
and internal control plasmid BOS-
b
-galactosidase. Twenty-four hours
after transfection, the medium was replaced to include bovine TSH
(Sigma, Dorset, UK) or recombinant human TSH (Genzyme, West
Malling, UK) as appropriate. Sixteen hours later, cells were lysed and
assayed for luciferase and galactosidase activities. Data are the mean 6
se of at least three separate experiments performed in triplicate.
Radiolabeled ligand binding studies were performed using mem-
branes extracted from COS cells transiently transfected with the receptor
expression vectors described above. Membranes were prepared as pre-
viously described (23), and protein was quantified by the Bradford
assay. Equal amounts (30–70
m
g) of protein were incubated for 2 h with
0.5 kilobecquerels [
125
I]TSH (RSR, Cardiff, UK) in NaCl-free assay buffer
with isotonicity maintained with 280 mmol/L sucrose (23) and in the
presence of increasing amounts of unlabeled bovine TSH (Sigma, Dorset,
UK). Radiolabeled TSH bound after washing was determined by
g
-counting.
Results
The results of thyroid function tests of all family members
are shown in Table 1. The biological activity of TSH in serum
from the proband was measured in Chinese hamster ovary
cells expressing recombinant human TSH receptor, and the
ratio of bioreactivity to immunoreactivity was normal at 0.69
(normal range, 0.6–2.1). This is in contrast to a recent report
of congenital hypothyroidism caused by biologically inactive
TSH due to a mutation in the TSH
b
-subunit gene (24). Thus,
the patient demonstrated features of TSH resistance, namely
reduced circulating thyroid hormone levels together with
elevated bioactive TSH. As the endocrine abnormality ap-
peared to be confined to the thyroid, analysis of the TSH
receptor gene was undertaken. Direct sequencing of the ex-
tracellular and transmembrane-coding regions of the TSH
receptor showed that the patient was heterozygous for a
CGG to CAG mutation in exon 4 corresponding to an argi-
nine to glutamine change at codon 109 (R109Q) and was
heterozygous for a second TGG to TAG mutation in exon 10
corresponding to a premature termination at codon 546
(W546X; data not shown). The mutations were verified by
repeated sequencing of both genomic DNA and receptor
cDNA isolated from the patients’ Epstein-Barr virus-trans-
formed lymphocytes. Exons 4 and 10 were amplified and
sequenced from genomic DNA isolated from each parent,
showing that mother was heterozygous for the W546X mu-
tation, and the father was heterozygous for the R109Q
mutation.
The functional consequences of each mutation were in-
vestigated using a method similar to that employed by
Sunthornthepvarakul and colleagues (19). Expression vec-
FIG. 1. Free T
4
(F) and TSH (Ç) concentrations of the patient in
relation to T
4
dose. Normal ranges for free T
4
(dotted area) and TSH
(cross-hatched area) are shown. T
4
therapy was discontinued at 24
months of age.
TABLE 1. Thyroid function test results at diagnosis
TSH
(mU/L)
Free T
4
(pmol/L)
Free T
3
(pmol/L)
TSH receptor
mutation
Patient
a
92 10 R109Q, W546X
Mother 1.5 16.3 8.1 W546X
Father 3.9 11.2 6.2 R109Q
Normal range 0.4–4.0 9–20 3–7.5
a
Normal range for a neonate: TSH, 0.4–4.0 mU/L; free T
4
, 12–28
pmol/L.
NOVEL MUTATIONS IN THE TSH RECEPTOR GENE 1095
tors encoding wild-type or mutant receptor were transiently
cotransfected with a reporter gene consisting of the glyco-
protein hormone
a
-subunit promoter and luciferase gene
(
a
LUC) into JEG3 cells. The
a
LUC reporter is highly respon-
sive in this system due to tandemly repeated cAMP response
elements between 2146 and 2111 bp of the
a
-subunit pro-
moter (25). Each mutant demonstrated impaired signal trans-
duction in response to TSH compared to that of the wild type
(Fig. 2a). The W546X mutant did not stimulate reporter gene
activity above basal levels, whereas the R109Q mutant
showed a right-shifted dose-response profile, such that max-
imal wild-type activity was achieved at higher concentra-
tions of TSH. Similar results, indicating impaired signal
transduction, were obtained using recombinant human TSH
(data not shown). To recapitulate the parental genotypes in
vitro, either mutant was cotransfected in equal amounts with
wild-type receptor. The resulting dose-response profiles did
not differ from that seen with wild-type receptor alone,
whereas cotransfection of both mutants reproduced the ac-
tivity of transfection of R109Q alone (Fig. 2b). Thus, neither
mutant was able to dominantly inhibit the activity of wild-
type receptor, nor was any positive cooperativity between
the mutants observed. Although constitutive activation of
the cAMP cascade by unliganded TSH receptor has been
described in COS cells (7, 9, 14), such basal activity of either
wild-type or mutant TSH receptors could not be detected in
this assay. This may be due to a limitation of the luciferase
reporter system, as previous characterization of mutant TSH
receptors using this assay in COS cells also failed to show
constitutive receptor activity (19).
Binding studies with radiolabeled ligand were undertaken
using the same receptor expression vectors transiently trans-
fected in COS-7 cells (Fig. 3). The R109Q mutant showed a
binding capacity of 1140 cpm, which is 60% that of wild-type
receptor (1900 cpm), and raised EC
50
(20 vs. 7mUofthe
bovine TSH preparation we used; data not shown), consis-
tent with reduced binding affinity for TSH. When experi-
ments were performed on whole cells, the binding to R109Q,
although higher than that on W546X or vector alone, was too
low to obtain a curve, indicating very poor surface expres-
sion of the mutant (data not shown). The W546X mutant
receptor showed negligible specific binding for TSH on ei-
ther whole cells or membranes, which is probably indicative
of very little surface expression of this receptor, although the
possibility that the truncated receptor still inserts into the
membrane but fails to bind TSH through lacking C-terminal
residues cannot be excluded. Nevertheless, poor cellular ex-
pression of prematurely truncated receptors is well de-
scribed (26–28).
Persistent elevation of serum TSH concentrations in this
child (Fig. 1) has prompted concern as to whether there may
be a risk of developing pituitary autonomy. Magnetic reso-
nance imaging (MRI) performed at 2 yr of age showed two
small areas of hypoattenuation after the administration of
gadolinium (Fig. 4). A T
3
suppression test was subsequently
performed, which showed brisk reduction of TSH into the
FIG. 2. Function of wild-type and mutant TSH receptors. a, Activation of
a
LUC by wild-type or mutant receptors transfected individually. Cells
were transfected with 500 ng reporter, 100 ng receptor expression vector, and 100 ng BOS-
b
-galactosidase. Luciferase activity was determined
after incubation with 0–100 mU/mL bovine TSH and normalized for transfection efficiency using
b
-galactosidase activity. The mean (6SE)
responses to bovine TSH are expressed relative to the maximum response obtained with wild-type receptor, which was approximately 10-fold.
b, Coexpression of wild type with each mutant receptor expression vector. Cells were transfected with reporter and reference plasmids, as
described in a, and 50 ng receptor expression vectors in the indicated combinations. For comparison 50 ng R109Q vector were transfected
individually with 50 ng pSVL to correct for DNA concentration. Hormone-dependent activation after incubation with 0–100 mU/mL bovine TSH
was determined as described above. Where values are less than 10% of the mean, error bars have been omitted for clarity. The combinations
of wild-type and each mutant receptor reflect the heterozygous nature of each parent (mother and father), whereas the combination of both
mutant receptors represents the compound state of the proband.
1096 CLIFTON-BLIGH ET AL.
JCE&M•1997
Vol 82 • No 4
normal range after the administration of T
3
(Table 2). Cre-
atine kinase, cholesterol, and triglyceride levels showed a
normal response to exogenous T
3
, although there was little
change in serum sex hormone-binding globulin or alkaline
phosphatase levels. Overall, these results suggest normal
pituitary and peripheral tissue responsiveness to T
3
in the
patient. Sequencing of the thyroid hormone receptor
b
gene
in the proband was also normal (data not shown).
Discussion
We have described a child with persistent hyperthyro-
tropinemia together with normal serum thyroid hormone
concentrations associated with two novel mutations in the
TSH receptor gene. When tested individually, each mutant
receptor showed impaired function in transfection studies.
Although the R109Q mutant receptor showed only mild im-
pairment, clinical data from the proband suggest that this
mutation results in markedly abnormal thyroid function. It
is possible that the limited dynamic range (10-fold) of the
transfection assay may underestimate the in vivo conse-
quences of the R109Q mutation. Neither mutant receptor
affected wild-type receptor function when coexpressed,
which is concordant with the observation that both parents
have normal thyroid function. Coexpression of both mutant
receptors results in some residual functional activity, which
correlates with the partial TSH resistance seen in the patient.
As expected from the autosomal recessive nature of this
disorder, neither mutant was able to dominantly inhibit the
function of wild-type receptor.
Our case is the second recorded example of loss of function
mutations in the TSH receptor gene (19). All cases described
hitherto have been euthyroid, indicating that TSH resistance
in each case is only partial, such that the elevated TSH levels
are able to stimulate adequate thyroid hormone secretion. In
contrast, the phenotype of more severe TSH resistance is
represented by the recessively inherited hyt/hyt hypothyroid
mouse (29). Here, fetal onset of profound hypothyroidism is
associated with greatly elevated TSH levels. The homozy-
gous mutant thyroid gland is hypoplastic and demonstrates
diminished follicular size and reduced colloid. Recently, a
point mutation in the TSH receptor gene was identified in
this mouse, corresponding to a Pro to Leu change at codon
FIG. 4. Pituitary MRI scan in the proband at 2 yr of age. Two areas of hypoattenuation after the administration of gadolinium are indicated
(arrows). The contours of the gland are normal.
TABLE 2. Thyroid function and indexes of hormone action during
aT
3
suppression test
Results Basal
T
3
,15
m
g for
3 days (% of basal)
T
3
,30
m
g for
3 days (% of basal)
Free T
4
(pmol/L) 12.0 11.2 (93) 9.8 (82)
Free T
3
(pmol/L) 6.4 9.9 (155) 10.3 (161)
TSH (mU/L) 134.8 56.4 (42) 9.5 (7)
Sex hormone-binding
globulin (nmol/L)
114 110 (96) 103 (90)
Alkaline phosphatase
(IU/L)
212 209 (99) 184 (87)
Cholesterol (mmol/L) 5.8 4.8 (83) 4.0 (69)
Creatine kinase (IU/L) 186 135 (73) 132 (71)
Triglyceride (mmol/L) 1.3 0.6 (46) 0.7 (54)
FIG. 3. A representative experiment showing binding of [
125
I]TSH to
membrane-associated TSH receptors. COS-7 cells were transfected
with the same receptor expression plasmids as in Fig. 2, and mem-
branes were incubated with [
125
I]TSH and 0–100 mU/mL unlabeled
bovine TSH. Mean (6SE) binding values of triplicate determinations,
expressed as counts per min, are shown.
NOVEL MUTATIONS IN THE TSH RECEPTOR GENE 1097
556 in the transmembrane region, which abolished TSH bind-
ing and response to TSH in vitro (30).
The locations of loss of function mutations in the TSH
receptor identified to date are shown in Fig. 5. Study of these
mutations has defined some important residues for TSH
binding and receptor function. The previous report indicated
that mutation of proline at codon 162 to alanine retained
some biological activity, whereas mutation of isoleucine at
codon 167 to asparagine virtually abolished signal transduc-
tion (19). A structural model for the hormone-binding site
has been proposed on the basis of similarity between the
extracellular domain of the TSH receptor and the crystal
structure of the ribonuclease inhibitor (31, 32). From this
model, the arginine at codon 109, which is mutated in our
case, may be expected to project into solution, contributing
to the TSH binding cavity. This hypothesis is confirmed by
studies indicating that mutation of this residue to glutamine
interferes with TSH binding. Both arginine at codon 109 and
tryptophan at codon 546 are conserved in the TSH receptor
from a number of species. Furthermore, tryptophan at codon
546 in the TSH receptor is conserved at homologous positions
in both the LH/CG and FSH receptors (33, 34). Curiously, in
the LH/CG receptor, the residue homologous to arginine 109
is glutamine. However, the transfected R109Q mutant TSH
receptor did not mediate signal transduction in response to
human LH (data not shown).
Our case contributes two more important findings. First,
it describes the only occurrence hitherto of a TSH receptor
with a premature termination codon within its serpentine
portion, resulting in a biologically inactive product. The
mother of the proband is thus effectively hemizygous for
functional TSH receptors, suggesting that only a single nor-
mal TSH receptor allele is required to sustain normal thyroid
function. Second, TSH levels in the proband are the highest
of those reported to date. Elevated serum TSH levels are
characteristic of TSH resistance and probably represent re-
setting of the pituitary threshold for TSH suppression by
thyroid hormones (19), although the mechanism remains
obscure. In keeping with this, there is no evidence for resis-
tance to thyroid hormones in our patient. The occurrence of
possible pituitary abnormalities on a MRI scan in our case in
the context of persistently high TSH levels was disturbing,
although the significance of these findings remains uncer-
tain. The evidence of brisk inhibition of serum TSH levels
FIG. 5. Location of loss of function mutations in the TSH receptor structure. Mutations of proline at codon 162, isoleucine at codon 167, and
proline at codon 556 have been identified previously (19, 30). The R109Q and W546X mutations are arrowed.
1098 CLIFTON-BLIGH ET AL.
JCE&M•1997
Vol 82 • No 4
during a T
3
suppression test mitigates against pituitary au-
tonomy. It is also encouraging that the secretion of other
anterior pituitary hormones, the
a
-subunit/TSH molar ratio,
and the growth pattern are all normal. Nevertheless, en-
largement of the sella has been shown to occur in long term
juvenile and untreated congenital hypothyroidism (35, 36),
which may be proportional to the degree of TSH elevation
(36). Moreover, thyrotrope hyperplasia is evident at autopsy
in long term primary hypothyroidism (37). It is known from
animal studies that prolonged hypothyroidism may ulti-
mately lead to thyrotrope neoplasia (38). We intend to follow
the pituitary changes closely, with further imaging at regular
intervals.
It is clear that activating mutations of the TSH receptor
increase both thyrocyte growth and function (39). In contrast,
impaired TSH receptor function has not yet been associated
with disordered thyroid development in humans. A role for
TSH in thyroid ontogeny is suggested by evidence that thy-
roid development is arrested in late gestation in mice ho-
mozygous for a knockout of the glycoprotein hormone
a
-subunit common to TSH, LH, and FSH (40), and that sim-
ilarly in humans, thyroid dysgenesis has been reported in
some cases of TSH deficiency caused by homozygous mu-
tations in TSH
b
-subunit gene (41, 42). By analogy with the
hyt/hyt hypothyroid mouse, we speculate that a combination
of severe loss of function TSH receptor mutations will be
found in some cases of thyroid dysgenesis. However, it is
also relevant to note that cases of congenital hypothyroidism
and TSH hyporesponsiveness have been described in which
the TSH receptor gene is reportedly normal (43, 44), sug-
gesting that the molecular basis of athyreosis is likely to be
heterogeneous. Partial TSH resistance with normal gland
development, low or normal thyroid hormone concentra-
tions, and elevated bioactive TSH levels may be a more
readily identifiable entity. The study of such cases will con-
tinue to provide valuable insights into the function of the
TSH receptor.
Acknowledgment
The authors are indebted to Prof. G. Vassart for providing the wild-
type TSH receptor cDNA cloned in pSVL.
Note Added In Proof
Since the submission of this manuscript, de Roux et al. have reported
compound heterozygosity for the W546X and another mutation in the
TSH receptor in a case of TSH resistance. N. de Roux, M. Misrahi, R.
Brauner, M. Houang, J. C. Carel, M. Granier, Y. Le Bouc, N. Ghinea, A.
Boumedienne, J. E. Toublanc, E. Milgrom. 1996 Four families with loss
of function mutations of the thyrotropin receptor. J Clin Endocrinol
Metab. 81:4229–4235.
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